Use of a cyclosporin for the treatment of hepatitis C infection and pharmaceutical composition comprising said cyclosporin

ABSTRACT

This invention relates to the use in the treatment of HCV infection, either as single active agents or in combination with another active agent, of a cyclosporin having increased cyclophilin binding activity and essentially lacking immunosuppressive activity.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.11/406,800, filed Apr. 12, 2006 now U.S. Pat. No. 7,439,227, which is anational phase application of international application PCT/IB05/02940designating the United States, filed on Oct. 3, 2005, which applicationclaims priority to international application PCT/IB04/03205, filed Oct.1, 2004.

The present invention relates to the use of a cyclosporin for thetreatment of hepatitis C virus (HCV) infection and to a pharmaceuticalcomposition comprising said cyclosporin.

HCV was cloned and characterized about 15 years ago by Choo andcolleagues (see Science 244, (1989), 359-362). HCV belongs to the familyFlaviviridae and comprises an enveloped nucleocapsid and asingle-stranded RNA genome of positive polarity (see Bartenschlager etal., Antiviral Res. 60, (2003), 91-102). HCV is transmitted primarily byblood, blood products and vertical transmission during pregnancy.Introduction of diagnostic tests for screening blood products hassignificantly reduced the rate of new infection.

Still, HCV remains a serious medical problem. There are currently about170 million people infected with HCV. The initial course of infection istypically mild. However, the immune system is often incapable ofclearing the virus, and people with persistent infections are at a highrisk for liver cirrhosis and hepatocellular carcinoma (see Poynard etal., Lancet 349, (1997), 825-832).

There is no vaccine available, and therapeutic options are very limited(see Manns et al., Indian J. Gastroenterol. 20 (Suppl. 1), (2001),C47-51; Tan et al., Nat. Rev. Drug Discov. 1, (2002), 867-881).

Current therapy is based on a combination of interferon alpha andribavirin. This therapy produces a sustained anti-viral response in85-90% of patients infected with genotypes 2 and 3, but, unfortunately,only in about 45% of patients infected with the prevalent genotype 1.Furthermore, side effects are significant and include myalgia,arthralgia, headache, fever, severe depression, leucopenia andhaemolytic anaemia.

Clearly, additional therapies, with a higher antiviral activity and abetter safety profile, are required for the treatment of HCV infection,particularly e.g. in the case of the prevention of HCV recurrence. Inorder to establish the safety profile, criteria such as low cytotoxicityand cytostatic and high selectivity index are particularly relevant forclinical treatment of HCV infection.

A novel approach for the treatment of HCV infection using cyclosporinswas recently described by clinical observations (see Teraoka et al.,Transplant Proc., 1988, 20 (3 suppl 3), 868-876, and Inoue et al. JGastroenterol, 2003, 38, 567-572). Recently it was shown thatCyclosporin A (CsA) inhibited the in vitro intracellular replication ofan HCV subgenomic replicon at clinically achievable drug concentrations(see Watashi et al., Hepatology 38, 2003, 1282-1288, and Nakagawa etal., BBRC 313, 2004, 42-47). Both groups suggested that the anti-HCVeffect of CsA was not associated with immunosuppressive activity basedon observations made with the use respectively of an immunosuppressivemacrolide, i.e. the compound known under the name FK 506 and anon-immunosuppressive Cyclosporin A derivative, i.e. the compound knownunder the name NIM 811 or [MeIle]⁴-CsA. Nakagawa et al. consider thatexpanding applications of CsA may cause substantial problems due to itswell-known immunosuppressive properties and suggest that one solution toovercome this problem would be to consider the use ofnon-immunosuppressive cyclosporin analogs.

During the last 15 years, a number of medicinal chemistry studies havebeen conducted with the aim to identify such non-immunosuppressivecyclosporin analogs and compound NIM 811 is one of the mostrepresentative compounds having such a property.

NIM 811, along with 9 other Cyclosporin A derivatives, were reported byKo et al. in patent application EP 0 4840 281 for theirnon-immunosuppressive properties and were considered as beingpotentially useful in the treatment of HIV infection and the preventionof AIDS. The design of those derivatives involved the modification ofthe amino-acids in 4- and/or 5-positions of Cyclosporin A.

By modifying amino-acids in 2- and/or 6-positions of Cyclosporin A,Sigal et al. synthesised a total of 61 cyclosporin analogs and observedthat such chemical modifications induce a decrease in theimmunosuppressive activity (see Sigal et al., J. Exp. Med., 173, 1991,619-628).

Further attempts for modifying amino-acid in 3-position of Cyclosporin Ain order to obtain non-immunosuppressive compounds were described inparticular by Barriére et al., in WO 98/28328, WO98/28329, and WO98/28330.

Wenger et al. have designed a series of compounds that differ fromCyclosporin A in position 3, in which they contain an N-methylated,nonbulky hydrophobic or neutral amino acid other than a glycine, and inposition 4, in which they contain an N-methylated or N-ethylatedhydrophobic or neutral amino acid other than a leucine and they reportthat those compounds have a high potency to inhibit HIV-1 replicationand essentially lack immunosuppressive activity (see Internationalpatent application WO 00/01715 and Tetrahedron Lett., 41, (2000),7193-6).

The aim of the present invention is to provide the clinician with a newtherapy for the treatment of HCV infection, particularly e.g. in thecase of the prevention of HCV recurrence. This therapy should offer ahigher antiviral activity and a better safety profile in comparison tothe already approved therapy or the newly proposed ones.

The present inventors surprisingly found that the administration to apatient infected with HCV of a very specific compound, i.e.[D-MeAla]³-[EtVal]⁴-CsA, meets the above requirements. They observedthat, in addition to its non-immunosuppressive property,[D-MeAla]³-[EtVal]⁴-CsA has a significantly increased affinity forcyclophilins, which increased affinity is correlated with an elevatedefficacy against inhibition of HCV replication.

Accordingly, one of the subject-matters of the present invention relatesto the use of [D-MeAla]³-[EtVal]⁴-CsA for the manufacture of a medicinalproduct intended for the treatment of HCV infection in a patient.

[D-MeAla]³-[EtVal]⁴-CsA has been reported by Wenger et al. in WO00/01715 and it has been attributed the CAS Registry Number 254435-95-5.It is a cyclic undecapeptide described by the following formula:

-MeBmt-αAbu-D-MeAla-EtVal-Val-MeLeu-Ala-(D)Ala-    1    2      3      4   5     6    7     8 MeLeu-MeLeu-MeVal-   9    10    11where MeBmt is N-methyl-(4R)-4-but-2E-en-1-yl-4-methyl-(L)threonine,αAbu is L-α-aminobutyric acid, D-MeAla is N-methyl-D-alinine, EtVal isN-ethyl-L-valine, Val is L-valine, MeLeu is N-methyl-L-leucine, Ala isL-alanine, (D)Ala is D-alanine, and MeVal is N-methyl-L-valine. Theconventional numbering of amino acid positions generally used inreference of Cyclosporin A is shown below the formula. This is achievedby using composite names comprising a first portion indicating theidentity of residues that are different from those in cyclosporin A andproviding their position, and a second portion labelled “CsA” indicatingthat all other residues are identical to those in Cyclosporin A. Forexample, [MeIle]⁴-CsA is a cyclosporin that is identical to cyclosporinA except that MeLeu in position 4 is replaced by MeIle(N-methyl-L-isoleucine).

The present invention will be explained further with followingexperiments and with the drawing in which:

FIG. 1 represents a dose response histogram measured by luciferase assayin infected Huh-7-Lunet cells;

FIG. 2 represents a dose response histogram measured by luciferase assayin infected Huh-7.5 cells;

FIG. 3 represents clearance response curves of infected Huh-9-13 cells;

FIG. 4 represents 3D representation of dose response with combinationIFN/[D-MeAla]³-[EtVal]⁴-CsA.

Current medical uses of Cyclosporin A relate to the ability of thiscompound to suppress the cell-mediated immune response by preventingproduction and release of several autocrine T-cell growth factors,including interleukin 2 (IL-2), from activated T cells (see Borel (1989)Transplant. Proceed. 21, 810-815; Kronke et al. (1984) Proc. Natl. Acad.Sci. USA 81, 5214-5218; Faulds et al. (1993) Drugs 45, 953-1040). Uponentry into cells, Cyclosporin A binds to cyclophilins with high affinity(see Handschumacher et al. (1984) Science 226, 544-547). As amongdifferent biological function, they have peptidyl-prolyl cis-transisomerase (PPlase) activity that can be measured in vitro (see Fischeret al. (1989) Nature 337, 476-478; Takahashi et al. (1989) Nature 337,473-475). Critical for the immunosuppressive effect of cyclosporin A isan interaction between cyclophilin-Cyclosporin A complex and calcium-and calmodulin-dependent serine/threonine phosphatase 2B (calcineurin)(see Hauske (1993) DN&P 6, 705-711, Friedman et al. (1991) Cell 66,799-806; Liu et al. (1991) Cell 66, 807-815). Formation of this ternarycomplex results in an inhibition of the phosphatase activity ofcalcineurin. (see Jain et al. (1993) Nature 365, 352-355; Rao et al.(1997) Annu. Rev. Immunol. 15, 707-747; Crabtree (1999) Cell 96,611-614). Calcineurin promotes the selective dephosphorylation of NF-ATthat then translocates to the nucleus where it associates with activatorprotein 1 and transactivates target genes, including the IL-2 gene.

It is believed that, due to the amino-acids in 3- and 4-positions,[D-MeAla]³-[EtVal]⁴-CsA has a dramatically reduced ability to interactwith calcineurin as shown by transcriptional and immunological assays aswell as a significantly increased affinity for cyclophilins as indicatedby assays of inhibition of peptidyl-prolyl cis-trans isomerase activity.

Peptidyl-prolyl cis-trans isomerase (PPlase) activity of cyclophilinswas determined using a procedure adapted from Kofron et al. (seeBiochemistry 30, 6127-6134 (1991); J. Am. Chem. Soc. 114, 2670-2675(1992)). N-succinylated Ala-Ala-Pro-Phe-para nitro-aniline(Suc-AAPF-pNA, Bachem, Bubendorf, Switzerland) was used as thesubstrate. The assay was based on the preferential chymotrypsin cleavageof the trans isoform of the Phe-pNA bond in the tetrapeptideAla-Ala-Pro-Phe-pNA. This cleavage liberates the para-nitroanilinemoiety that can be detected and quantitated at 390 nm (ε=11,814M⁻¹cm⁻¹). Schutkowski et al. (1995) Biochemistry 34, 13016-13026.Cis-trans isomerization is catalysed by cyclophilin (PPlase, EC5.2.1.8). After mixing CsA or another cyclosporin (10⁻⁹−2×10⁻⁵ M finalconcentrations prepared from 1000-fold concentrated stock solutions inethanol) with 0.1 μg cyclophilin (Sigma) in a total volume of 1.5 ml of40 mM Hepes, pH 7.9, and incubation for 50 min on ice, the reactionmixture was transferred to a cuvette that was kept at 10° C. in a Varianspectrophotometer (Varian). Subsequent to the addition of 3.75 mg ofchymotrypsin (70 μl of a solution of chymotrypsin in 10 mM HCl), thereaction was initiated by addition of 10 μl of a 3.2 mM solution ofSuc-AAPF-pNA in 0.5 M LiCl/trifluoroethanol. The reaction was monitoredfor 3 min, and an initial rate constant was determined from the dataobtained. As a control, an initial rate constant was also determined fora parallel reaction that lacked cyclophilin. Concentration-responsecurves were established for cyclosporin A and other cyclosporins, andIC₅₀ (50% inhibitory concentration) values of different cyclosporinswere expressed relative to that of cyclosporin A (1.0). A value lessthan 1 means that the compound has an higher cyclophilin affinity thanCsA.

A NF-AT-dependent reporter assay was used initially to estimateimmunosuppressive activities of cyclosporins. Baumann et al. (1992)Transplant. Proc. 24, 43-48. Jurkat T cells stably transfected with areporter construct containing a bacterial β-galactosidase gene under thecontrol of a promoter of an IL-2 gene were obtained from G. Zenke,Novartis Pharma AG, Basel, Switzerland. The cells were grown in RPMI1640medium supplemented with 10% heat-inactivated fetal calf serum, 100 u/mlpenicillin, 100 μg/ml streptomycin, 2 mM glutamine, 50 μM2-mercaptoethanol and 100 u/ml hygromycin B. The cells were stimulatedby the addition of 2.4 μM phorbol-2-myristate-13-acetate and 75 μg/mlphytohemagglutinin in the presence or absence of cyclosporin A oranother cyclosporin (10⁻⁹−2×10⁻⁵ M final concentrations prepared from1000-fold concentrated stock solutions in ethanol). Subsequent toincubation for 20 h at 37° C., cells were harvested and lysed in 50 mMNa₂HPO₄ (pH 9.0), 10 mM KCl, 1 mM MgSO₄, 1% Triton X-100, 0.5 mM4-methylumbelliferyl-β-D-galactoside (Sigma, Buchs, Switzerland). Theβ-galactosidase reaction was allowed to proceed for 1 h in the dark atroom temperature. Fluorescent 4-methyl-umbelliferone was assayedfluorometrically in the supernatant solution (excitation: 355 nm;emission: 460 nm). Concentration-response curves were established forcyclosporin A and other cyclosporins, and the IC₅₀ values of differentcyclosporins were calculated relative to that of cyclosporin A (1.0). Avalue higher than 1 means that the compound is less immunosuppressivethan CsA.

Example results are shown in Table 1 below.

TABLE 1 Cyclophilin binding (PPlase) and immunosuppressive (IL-2)activities of CsA and other cyclosporins Compound PPlase IL-2 CsA 1.01.0 [D-MeAla]³-[EtVal]⁴-CsA 0.3 7161 [MeIle]⁴-CsA 0.5 2250The data shown in Table 1 revealed that certain substitutions inposition 4 (i.e., Val, Ile) dramatically reduced immunosuppressiveactivity (measured as inhibition of IL-2 expression) as well asdetectably enhanced cyclophilin binding activity (measured as inhibitionof PPlase activity of cyclophilin). Substitution in position 3 resultedin a substantial further increase in cyclophilin binding activity (by 2fold or more; cf [D-MeAla]³-[EtVal]⁴-CsA. It had a higher cyclophilinbinding activity and a lower residual immunosuppressive activity than[MeIle]⁴-CsA, the best reference compound available from the literature.[MeIle]⁴-CsA is also known as NIM811.Non-immunosuppressive Activity of [D-MeAla]³-[EtVal]⁴-CsA

In a confirmatory analysis, immunosuppressive activities of CsA,[MeIle]⁴-CsA and [D-MeAla]³-[EtVal]⁴-CsA were estimated using the mixedlymphocyte reaction. In this assay, cyclosporins were dissolved inethanol (10 mg/ml). Freshly isolated CD4⁺ PBMCs from two healthy donorswere mixed subsequent to inactivation by irradiation of one of thepopulations (stimulator cells; S). After five days of co-culture in thepresence or absence of a cyclosporin (1 μg/ml), the proliferativeresponse of the non-inactivated cell population (responder cells; R) wasdetermined by [³H]-thymidine incorporation.

The assay was conducted reciprocally with the two cell populations, eachbeing inactivated and stimulated in turn. Stimulation (%) of respondercells was calculated by the formula:Percent stimulation=100×(sample with cyclosporin−background)/(samplewithout cyclosporin−background)

Sample refers to a mixture of stimulator and responder cells. Backgroundrepresents a control in which only stimulator cells are mixed. Resultsare shown in Table 2. They were interpreted to mean that both[MeIle]⁴-CsA and [D-MeAla]³-[EtVal]⁴-CsA are essentially devoid ofimmunosuppressive activity.

TABLE 2 Proliferative response of CD4⁺ PBMCs in the presence/absence ofcyclosporins % Stimulation Standard Co-culture Compound N (relative)deviation R1 × S2 None 8 100 30 R1 × S2 CsA 4 29 3 R1 × S2 [MeIle]⁴-CsA4 84 13 R1 × S2 [D-MeAla]³-[EtVal]⁴-CsA 4 75 11 R2 × S1 None 8 100 9 R2× S1 CsA 4 9 2 R2 × S1 [MeIle]⁴-CsA 4 75 9 R2 × S1[D-MeAla]³-[EtVal]⁴-CsA 4 65 7 S1 × S2 None 4 0 0.6

R1×S2 refers to a co-culture of responder cells from donor 1 andstimulator cells from donor 2. N is the number of measurements.

High Anti-HCV Activity and Low Cytotoxicity/Cytostatic Effect of[D-MeAla]³-[EtVal]⁴-CsA

As mentioned previously, infection with hepatitis virus C(HCV) is aserious health problem because persistently infected patients are at ahigh risk for developing chronic liver diseases including cirrhosis andhepatocellular carcinoma. Current available therapy is inadequate for alarge fraction of the latter population as well as is associated withsignificant side effects. Until recently, development of more effectivetherapies was hindered by the absence of an appropriate in vitro modelof HCV replication that allows screening of potentially active compoundsprior to evaluation in human clinical trials. This obstacle was overcomeby the development of genetically modified HCV minigenomes (replicons)that self-amplify in cultured hepatoma cells to high levels (Lohmann etal. Science 285, (1999), 110-113). This HCV replicon system has rapidlybecome the standard tool for studying HCV replication, pathogenesis andpersistence (Bartenschlager et al. Antiviral Res. 60, (2003), 91-102).The HCV genome consists of a single-stranded RNA that contains a singleopen reading frame for a polyprotein of about 3000 amino acids.Translation of this polyprotein is initiated at an internal ribosomeentry site (IRES) located at the 5′ end of the RNA. The HCV polyproteinis cleaved into at least ten proteins. They include capsid protein C,envelope proteins E1 and E2, possible viroporin protein p7,non-structural proteins NS2 and NS3 having serine proteinase as well asATPase/helicase activities, NS4A, membraneous web-inducing protein NS4B,NS5A and RNA-dependent RNA polymerase NS5B. The first successfulreplicon was a bicistronic RNA containing in a 5′ to 3′ direction an HCVIRES, a coding sequence for a neomycin phosphotransferase, an IRES froman encephalocarditis virus and coding sequences for HCV proteins NS3 toNS5. Subsequent to introduction into Huh-7 cells and selection usingG418 (geneticin), this replicon could be shown to replicate autonomouslyto high levels (1,000-5,000 copies/cell) (Lohmann et al., 1999).Characterization of the system revealed that replication efficiencydepended on permissiveness of the host cell and, importantly, on theselection of cell culture-adaptive mutations in the HCV protein-codingsequences. Replication was found to be sensitive to interferon alpha,providing evidence for the relevance of the system for screening drugsthat have in vivo efficacy. Variant replicons were also constructed inwhich the neomycin phosphotransferase-coding sequence was replaced,e.g., by a luciferase-coding sequence or by sequences coding for aluciferase-ubiquitin-neomycin phosphotransferase fusion protein.Replication of the latter variant replicons can be assayed by theconvenient luciferase assay, whereas replication of the former repliconrequires determinations of RNA copy number.

Watashi et al. (2003) demonstrated by Northern blot and quantitativeRT-PCR (reverse transcriptase polymerase chain reaction) that HCV RNAaccumulation was inhibited by CsA but not by the immunosuppressivemacrolide FK506 and the non-immunosuppressive CsA derivative PSC 833 inHCV replicon-containing MH-14 cells. Their assays that involved 7-dayexposures of cells to active agents revealed that HCV RNA titer wasreduced by about 200 fold in the presence of 1 μg/ml cyclosporin A. Theyfurther found that non-immuno-suppressive cyclosporin [MeIle]⁴-CsA alsoinhibited HCV replication. Results indicated that [MeIle]⁴-CsA was aboutequally as effective as CsA in reducing HCV RNA titer.

To determine whether the cyclosporin of the present invention hasanti-HCV activity and, should it has such activity, how this activitycompares with the activities of CsA and [MeIle]4-CsA, experiments werecarried out that compared inhibitory effects of CsA, [MeIle]⁴-CsA and[D-MeAla]³-[EtVal]⁴-CsA in HCV replicon systems.

Assays used Huh 5-2 cells that contained a bicistronic RNA encoding afirefly luciferase-ubiquitin-neomycin phosphotransferase fusion proteinand HCV proteins NS3-5. The viral sequences originated from an HCV virusof genotype 1b. Cells were cultured in RPMI 1640 medium (Gibco)supplemented with 10% fetal calf serum, 2 mM glutamine (LifeTechnologies), 1× non-essential amino acids (Life Technologies), 100u/ml penicillin, 100 μg/ml streptomycin and 250 μg/ml G418 (Geneticin,Life Technologies) at 37° C. and 5% CO₂. For antiviral (replication)assays, cells were seeded at a density of 7000 cells/well in 96-wellView Plates™ (Packard) in the same medium except for G418. After a 24-hincubation, medium was removed, serial dilutions of test compounds inmedium were added, and cells were incubated for an additional 72 h.

Antiviral effects were estimated either by luciferase assay orquantitative RT-PCR. To carry out luciferase assays, medium was removed,and cells were washed with PBS. Subsequent to lysis in 50 μl ofGlo-lysis buffer (Promega) for 15 min, 50 μl of Stead-Glo LuciferaseAssay Reagent (Promega) were added to cell lysates. Luciferase activitywas measured using a luminometer, and the signal from each test well wasexpressed as a percentage of the signal measured in wells of culturesnot exposed to a test compound.

Cell density and cytostatic effects were estimated in parallel culturesin regular 96-well plates (Beckton-Dickinson) using the MTT assay(CellTiter 96^(R) AQ_(ueous) Non-Radioactive Cell Proliferation Assay,Promega). In this assay,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) is bioreduced to a formazan that is quantitated at 498 nm in aplate reader. Formazan production is directly correlated with number oflife cells.

RT-PCR analysis quantitated the neomycin region of replicons using anABI PRISM 7700 sequence detector (Applied Biosystems, Foster City,Calif.). The forward and reverse primers used were5′-CCGGCTACCTGCCCATTC-3′ and 5′-CCAGATCATCCTGATCGACAAG-3′, respectivelySEQ ID: 1, SEQ ID NO: 2. The fluorogenic probe was5′-ACATCGCATCGAGCGAGCACGTAC-3′ SEQ ID No: 3. As an internal control, aplasmid containing part of the neomycin phosphotransferase gene sequencewas used.

Results from these experiments permitted calculation of EC₅₀ for thedifferent cyclosporins, which is the effective concentration required toinhibit HCV replicon replication by 50%, and of CC₅₀, which is theconcentration required that inhibits the proliferation of exponentiallygrowing cells by 50%, and a selectivity index SI, which is the ratiobetween CC₅₀ and EC₅₀.

Table 3 shows values obtained from Huh 5-2 cells using luciferaseactivity assays for estimation of replication efficiency and MTT assaysfor calibration of luciferase assays and for estimation of cytostaticeffects of compounds. In agreement with the above-discussed observationsby Watashi et al. (2003), CsA and [MeIle]⁴-CsA had similar anti-HCV(replication) activities.

Surprisingly, [D-MeAla]³-[EtVal]4-CsA was considerably more potent thanCsA and [MeIle]⁴-CsA. It was also noted that the 50% cytostaticconcentration (CC50) for [D-MeAla]³-[EtVal]4-CsA was significantlyhigher than the values determined for CsA and [MeIle]⁴-CsA.Consequently, a considerably higher selectivity index was found for[D-MeAla]³-[EtVal]⁴-CsA as compared to the two other cyclosporins.Analogous experiments in which EC₅₀ values were derived fromdeterminations of RNA titers using quantitative RT-PCR yielded similarconclusions. SI values of 45*, 73 and 625* were obtained for CsA,[MeIle]⁴-CsA and [D-MeAla]³-[EtVal]⁴-CsA, respectively. Asterisksindicate that the lower of two independently determined values arepresented.

TABLE 3 EC₅₀, CC₅₀ and SI values determined from luciferase assays ofHCV RNA replication and MTT assays of cytotoxicity in Huh 5-2 cellscomprising a luciferase-containing HCV minireplicon CC₅₀ EC₅₀ (μg/ml)+/− (μg/ml) +/− Selectivity Compound Std. Dev. Std. Dev. index CsA 0.28+/− 0.13 11.6 +/− 5.6 41 [D-MeAla]³-[EtVal]⁴-CsA 0.03 +/− 0.04 >27 >900[MeIle]⁴-CsA 0.22 14 64Antiviral Activity of [D-MeAla]³-[EtVal]⁴-CsA Measured in InfectedTarget Cells with Recombinant HCV

The anti-HCV activity of [D-MeAla]3-[EtVal]4-CsA compared to CsA wasfurther determined in culture systems approaching the in vivo situation.The method used hepatoma cells that had been infected with an infectiousfull length chimeric HCV construct or the same virus that was modifiedto carry a luciferase receptor gene. After the treatment of the infectedcells with the cyclosporin of the invention or CsA, the luciferaseactivity was measured as being directly correlated to the inhibition ofthe viral replication.

Infectious HCV viruses of full-length chimeric genome between HCVstrains J6 and JFH1 (Jc1) were used to inoculate the hepatoma cells ofthe assays. The construct of Jc1 virus was also modified to obtain abicistronic genome carrying a luciferase reporter gene (Jc1-Luc).Twenty-four and ninety-six hours after the transfection of RNAtranscripts of the genomes by electroporation of Huh-7.5 cells, cellculture supernatant was collected. Supernatants were filtered (0.45 μM)and cell culture infectious dose 50 (CCID50) per ml were determined bythe limiting dilution assays according to Lindenbach et al. (Science,309, (2005), 623-626). The CCID50 were 1.3×105 for Jc1 and 4.2×103 forJc1-Luc.

Assays used either Huh-7-Lunet or Huh-7.5 cells (Lohmann et al., Science285(5424), (1999), 110-113). Cells were grown in Dulbecco's modifiedEagle's Medium (DMEM; Gibco) supplemented with 10% heat-inactivatedfetal bovine serum (FCS) (Integro), 1× non-essential amino acids(Gibco), 100 IU/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) or25 μg/ml hygromycin (Gibco) for Huh-mono cells at 37° C. and 5% CO2. Forantiviral (replication) assays, Huh-7-Lunet and Huh-7.5 cells wereseeded at a density of 2×104 or 4×104 cells per well of a 12-well plate.Twenty four hours later, the medium was replaced by 0.5 ml of theJc1-Luc virus stock (12-well plates) or 0.25 ml of the Jc1 virus stock(12-well plates). Four hours later, the virus inoculum was replaced bymedium containing different concentrations of CsA or[D-MeAla]3-[EtVal]4-CsA and were further incubated for an additional 72hours.

The inhibition of viral replication were estimated by luciferase assay.To carry out luciferase assay, cells were harvested, washed with PBS andlysed in luciferase lysis buffer (1% Triton X-100, 25 mM glycylglycine,15 mM MgSO4, 4 mM EGTA, and 1 mM DTT). Firefly luciferase activity wasmeasured according to Krieger et al. (J Virol, 75(10), (2001),4614-4624). Briefly, after one freeze/thaw cycle, cells were resuspendedand 100 μl of cell lysate was mixed with 360 μl assay buffer (25 mMglycylglycine, 15 mM MgSO4, 1 mM DTT, 2 mM ATP, 15 mM potassiumphosphate buffer, pH 7.8) and 200 μl substrate solution (200 mMluciferin, 25 mM glycylglycine). Finally, luminescence was measured byusing a Lumat LB9507 luminometer (Berthold) for 20 samples.

In these examples (FIGS. 1 and 2), both [D-MeAla]³-[EtVal]⁴-CsA (whitebars) and CsA (black bars) resulted in a dose-dependent antiviralactivity, whereby [D-MeAla]³-[EtVal]⁴-CsA proved again superior overCsA, thus corroborating the data obtained with the subgenomic replicons.A 10-fold higher concentration of CsA was needed to result in the samereplication inhibiting effect as the cyclosporin of the invention.

High Affinity of the Cyclosporin of the Invention for Cyclophilin

In the above-discussed observations by Watashi et al. (2003) andNagakawa et al. (2003), the anti-HCV effect was related to the bindingcapacity of cyclosporins to the cyclophilins. The effects on the PPlaseactivity of CsA, [MeIle]⁴-CsA and [D-MeAla]³-[EtVal]⁴-CsA was measuredfor cyclophilins to determine the more potent inhibitor of PPlaseactivity of cyclophilin, e.g. cyclophilin A, and, consequently, of HCVreplication.

Commercial human recombinant cyclophilin A (Sigma) was used in theassays. PPlase activity of cyclophilins was determined using achymotrypsin-coupled spectrophotometric assay according toGarcia-Echverria et al. (BBRC, 191, (1993), 70-75). This method is basedon the high trans selectivity of chymotrypsin for peptides of the typeN-succinyl-ala-ala-pro-phe-p-nitroanilide. The peptides cleavageliberated the para-nitroaniline moiety that could be detected andquantitated at 390 nm. The hydrolysis of the cis form was limited by therate of cis-trans isomerisation carried out by cyclophilin A. Thepeptide was made up in a solution of 25 nM LiCl in2,2,2-trifluoroethanol at 470 mM to enhance the cis conformer peptidespopulation. The assay was performed on the split beam spectrophotometerand the water bath was set at 5° C. Cyclophilin A (7500 μmol/mg totalenzyme concentration; Sigma) were dissolved at 20 nM in a buffer (35 mMHEPES and 0.26 mg/ml chymotrypsin (specific activity 50 units/mg), pH7.8 with KOH) and were incubated for 6 minutes at room temperaturefollowed by 54 minutes in the water bath. CsA, [MeIle]⁴-CsA or[D-MeAla]³-[EtVal]⁴-CsA were added as appropriate in these incubationsusing a concentration range of 2-50 nM. Then 3.5 ml of the incubatedcyclophilin was added to the sample cuvette. The reference cuvettecontained a reaction that had gone to completion to balance thereference beam. Peptide was added at 25 μM to initiate the reaction andthe change in absorbance was monitored at 10 data points per second. Asa control, rates were also determined for a parallel reaction thatlacked cyclophilin. These blank rates of peptide hydrolysis (i.e. in theabsence of cyclophilin) were subtracted from rates in the presence ofcyclophilin A.

The initial rates obtained from the PPlase assays were analysed by firstorder regression analysis by using first order transformation of thetraces of the time course of the change in the absorbance at 390 nm.Total enzyme concentration (E_(t)), the inhibitor dissociation constant(K_(i)) and the rate constant for the rate limiting reaction wascalculated with the software FigSyS (2003, Biosoft) by fitting the dataobtained from the regression analysis in the tight binding inhibitormultiprotein equation.

The tight binding inhibitor multiprotein equation had the followingformula:v=k*E _(t) *P-k*(-b-sqrt(b*b-4*c))/2where b is defined as b=−(E_(t)*P+l+K_(i)) and c is c=Et*P*I.Once Et, K_(i) and k were calculated by the computer for a given set ofdata, a graphic representation of the data was plotted and the linefitted to the points assuming tight inhibitor binding to a singleprotein, defined by the following equations:v=K*E _(t) *P-K(B-sqrt(B*B-4*C))/2where B=E_(t)*P+I+K_(i) and C=E_(t)*P*I.

TABLE 4 E_(t), K_(i) and k values of cyclophilin A for CsA, [MeIle]⁴-CsAand [D-MeAla]³-[EtVal]⁴-CsA determined from PPlase activity assays.Compound E_(t) (pmol/mg) K_(i) (nM) k (s − 1) CsA 7500 9.79 ± 1.37 0.17± 0.0069 [MeIle]⁴-CsA 7500 2.11 ± 0.32 0.17 ± 0.0068[D-MeAla]³-[EtVal]⁴-CsA 7500 0.34 ± 0.12 0.16 ± 0.0074

The lowest K_(i) of cyclophilin A observed for the cyclosporin of theinvention corroborated the high potent of antiviral activity, thespecificity and selectivity index (as above-mentioned) compared to CsAand [MeIle]⁴-CsA. Surprisingly, the non-immunosuppressive[D-MeAla]³-[EtVal]⁴-CsA showed an almost 6-fold higher affinity for thecyclophilin of the example compared to the other non-immunosuppressivecyclosporin [MeIle]4-CsA.

The above-described experimentation provided that[D-MeAla]³-[EtVal]⁴-CsA was a more effective inhibitor of HCVreplication than any other tested cyclosporin. This increased anti-HCVactivity correlated with the increased cyclophilin binding activity of[D-MeAla]³-[EtVal]⁴-CsA.

HCV Replicon Clearance and Rebound

The recurrence of HCV infection is a major problem of the diseaseespecially even with the use of potential efficient treatment, e.g.cyclosporin and/or Interferon. To study whether the more potent anti-HCVactivity of the cyclosporin of the invention as compared to CsA isreflected in the ability of the compound to more efficiently cure cellsproducing HCV replicon from those, an in vitro cell assay was performedbased on presence of the selective drug G418 for recombinant producedreplicon.

Assays used Huh-9-13 cells, human hepatoma cells (Huh-7) (Lohmann etal., Science 285(5424), (1999), 110-113) Cells were grown in the usualcomplete medium DMEM without G418 pressure. The cells were cultured inthe presence of either CsA or [D-MeAla]³-[EtVal]⁴-CsA (both at 0.5 or 1μg/ml) or were left untreated for 7 consecutive passages. Control wasperformed to guarantee that the absence of the G418 selective pressurewould not influence the HCV replicon content during several passages. Toconfirm that Huh-9-13 cells that had been treated for 7 days with[D-MeAla]³-[EtVal]⁴-CsA were indeed cleared from their replicon, G418selection (1000 μg/ml) was restarted for 2 more passages. Only thosecells that were still carrying the HCV replicon have been able toproliferate under these conditions and cells without replicon have diedin the presence of G418 during the rebound phase.

RT-PCR were performed on extracts of viral RNA of samples taken atdifferent passage points. The forward and reverse primers used were5′-CCGGCTACCTGCCCATTC-3′ and 5′-CCAGATCATCCTGATCGACAAG-3′, respectively.The fluorogenic probe was 5′-ACATCGCATCGAGCGAGCACGTAC-3′. As an internalcontrol, a plasmid containing part of the neomycin phosphotransferasegene sequence was used. Results were analysed and expressed as aquantity of replicon RNA (ng) per 1,000 cells and used to draft a graph.

Results from these experiments (FIG. 3) showed the superior antiviraleffect of [D-MeAla]³-[EtVal]⁴-CsA compared to CsA in this standard invitro cell assay. Surprisingly, the cyclosporin of the invention showedvirucidal effect and not only virustatic effect as the otherimmunosuppressive CsA. Indeed, when the [D-MeAla]³-[EtVal]⁴-CsA treatedHuh-9-13 cells (circles and square in FIG. 3) were again cultured in thepresence of G418 (rebound phase), the cultures died compared to CsAtreated cells (diamond and triangle). Both cultures that had beentreated with CsA for 7 consecutive passages were able to proliferate inthe presence of G418. This confirmed that [D-MeAla]³-[EtVal]⁴-CsA wasable to cure Huh-9-13 cells from their HCV replicon.

Drug Combination

Interferon (IFN) is part of the current therapy of HCV infection. Theeffect of [D-MeAla]3-[EtVal]4-CsA/IFN-α2a combination was evaluatedusing the method of Prichard and Shipman (Antiviral Res, 1990, 14,181-205). In brief, the theoretical additive effect is calculated fromthe dose-response curves of individual compounds by the equation offormula:Z=X+Y(1-X),where X represents the inhibition produced by [D-MeAla]3-(EtVal)4-CsAalone and Y represents IFN-α2a alone. Z represents the effect producedby the combination of [D-MeAla]3-[EtVal]4-CsA with IFN-α2a. Thetheoretical additive surface is subtracted from the actual experimentalsurface, resulting in a horizontal surface that equals the zero planewhen the combination is additive, a surface that lies above the zeroplane indicates a synergistic effect of the combination and a surfacebelow the zero plane indicates antagonism. The antiviral assay wascarried out essentially as described above for Huh 5-2 cells except thatcompounds were added in checkerboard format. For each compound threereplicate plates were used to measure the dose response curve of eachindividual compound. The data obtained from all three plates were usedto calculate the theoretical additive surface. Combination studies foreach pair of compounds were also done in triplicate. Data were analysedfor variance by the ANOVA test.

A slight synergistic activity was noted at the highest concentrations ofIFN-α2a used, but overall the combined anti-HCV activity of[D-MeAla]3-[EtVal]4-CsA with IFN-α2a can be considered as additive (FIG.4).

The findings with [D-MeAla]³-[EtVal]⁴-CsA can be summarized as follows:

-   -   [D-MeAla]3-[EtVal]4-CsA has a more potent anti-HCV activity and        is less cytotoxic than CsA, as shown in an HCV subgenomic        replicon system.    -   This has been confirmed in an hepatoma cell culture infected        with a full-length infectious chimeric genome between HCV        strains J6 and JFH1.

[D-MeAla]3-[EtVal]4-CsA is able to cure cells from their HCV repliconmore efficiently than CsA

-   -   These effects are related to a more pronounced cyclophilin        binding affinity.    -   The anti-HCV activity of the combination        [D-MeAla]3-[EtVal]4-CsA/IFN-α2a is additive.

[D-MeAla]3-[EtVal]4-CsA can be used to treat patients infected with HCV.The active compound may be administered by any conventional route. Itmay be administered parentally, e.g., in the form of injectablesolutions or suspensions, or in the form of injectable depositformulations. Preferably, it will be administered orally in the form ofsolutions or suspensions for drinking, tablets or capsules.Pharmaceutical compositions for oral administration comprising acyclosporin of the invention are described in Examples. As isdemonstrated by the examples, such pharmaceutical compositions typicallycomprise a cyclosporin of the invention and one or more pharmaceuticallyacceptable carrier substances. Typically, these compositions areconcentrated and need to be combined with an appropriate diluent, e.g.,water, prior to administration. Pharmaceutical compositions forparenteral administration typically also include one or more excipients.Optional excipients include an isotonic agent, a buffer or otherpH-controlling agent, and a preservative. These excipients may be addedfor maintenance of the composition and for the attainment of preferredranges of pH (about 6.5-7.5) and osmolarity (about 300 mosm/L).

Additional examples of cyclosporin formulations for oral administrationcan be found in U.S. Pat. Nos. 5,525,590 and 5,639,724, and U.S. Pat.Appl. 2003/0104992. By the oral route, the indicated dosage of acyclosporin of the invention for daily to trice weekly administrationmay be from about 1 mg/kg to about 100 mg/kg, preferably from about 1mg/kg to about 20 mg/kg. By the intravenous route, the indicatedcorresponding dosage may be from about 1 mg/kg to about 50 mg/kg,preferably from about 1 mg/kg to about 25 mg/kg. An effective amount ofa cyclosporin of the invention is understood to be an amount that whenadministered repeatedly in the course of a therapeutic regimen to apatient in need of treatment of HCV infection results in an objectiveclinical response such as a statistically significant reduction in serumHCV titer or a significant reduction of serum ALT activity in thepatient.

Initial phase I clinical studies were carried out to assess the safetyof oral doses of [D-MeAla]³-[EtVal]⁴-CsA, and to determine thepharmacokinetic profile and safety profile of the drug substance.Studies showed that doses of 50 to 1600 mg in a micro-emulsion in waterwere well tolerated. Mild and short-lived side effects were observedincluding nausea, vomiting, abdominal pain, mild headaches. These sideeffects were not dose-related.

Numerous factors will be taken into consideration by a clinician whendetermining trial doses for testing efficacy of a pharmaceuticalcomposition comprising a cyclosporin of the present invention againstHCV infection. Primary among these are the toxicity and half-life of thechosen cyclosporin of the invention. Additional factors include the sizeof the patient, the age of the patient, the general condition of thepatient (including significant systemic or major illnesses includingdecompensated liver disease, severe preexisting bone marrow compromiseand other viral infections), the stage of HCV infection (acute vs.chronic) as indicated, e.g., by serum alanine aminotransferase (ALT)levels, the particular genotype of HCV, previous therapy of HCVinfection, the presence of other drugs in the patient, and the like. Acourse of treatment will require repeated administration of apharmaceutical composition of the invention. Typically, an adequate drugdose will be administered 3-7 times per week, and duration of treatmentmay be from about 4 weeks to 6 months, preferably from about 4 weeks toabout 12 months. Treatment may be followed by determinations of HCV inserum and measurement of serum ALT levels. The endpoint of treatment isa virological response, i.e., the absence of HCV at the end of atreatment course, several months after initiation of treatment, orseveral months after completion of treatment. HCV in serum may bemeasured at the RNA level by methods such as quantitative RT-PCR ornorthern blots or at the protein level by enzyme immunoassay or enhancedchemiluminescence immuoassay of viral proteins. The endpoint may alsoinclude a determination of a serum ALT level in the normal range.

A pharmaceutical composition of the present invention may comprise oneor more other ingredients active against HCV infection in addition to acyclosporin of the present invention such as, for example, anotherantiviral drug substance, e.g., ribavirin, or an interferon alpha. Acyclosporin of the invention and such other active ingredient can beadministered together as part of the same pharmaceutical composition orcan be administered separately as part of an appropriate dose regimendesigned to obtain the benefits of the combination therapy. Theappropriate dose regimen, the amount of each dose administered, andspecific intervals between doses of each active agent will depend uponthe specific combination of active agents employed, the condition of thepatient being treated, and other factors discussed in the previoussection. Such additional active ingredients will generally beadministered in amounts less than or equal to those for which they areeffective as single therapeutic agents. The FDA approved dosages forsuch active agents that have received FDA approval for administration tohumans are publicly available.

All patents, patent applications and publications cited herein shall beconsidered to have been incorporated by reference in their entirety.

The invention is further elaborated by the following examples. Theexamples are provided for purposes of illustration to a person skilledin the art, and are not intended to be limiting the scope of theinvention as described in the claims. Thus, the invention should not beconstrued as being limited to the examples provided, but should beconstrued to encompass any and all variations that become evident as aresult of the teaching provided herein.

EXAMPLE 1 Synthesis of [D-MeAla]³-[EtVal]⁴-CsA

(Translated from a Ph.D. thesis by Jean Francois Guichou entitled “Denouveaux analogues de Cyclosporin A comme agent anti-VIH-1”, Faculte desSciences, University of Lausanne, CH-1015 Lausanne, Switzerland (2001)).

Synthesis ofH-MeLeu-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(Oac)-Abu-Sar-OMe

4-Dimethylaminopyridine (DMAP) (41.5 mmoles; 5.8 g) was added to asolution of cyclosporin A (CsA) (8.3 mmoles; 10 g) in 100 ml aceticanhydride. The solution was stirred for 18 h at room temperature. Thereaction mixture was then diluted with 600 ml ethyl acetate, and washedtwice with water and four times with a saturated aqueous solution ofsodium bicarbonate. The organic phase was dried over anhydrous Na₂SO₄,filtered and solvent was evaporated under reduced pressure. The yellowresidue obtained was chromatographed on silica gel (eluent: 98:2dichloromethane/methanol) and recrystallized in ether. 9.5 g ofMeBmt(OAc)-CsA, a white powder, were recovered, representing a yield of92%.

Trimethyloxonium tetrafluoroborate (22.5 mmoles; 3.3 g) was added to asolution of MeBmt(OAc)-Cs (7.5 mmoles; 9.4 g) in 60 ml dichloromethane.After 16 h at room temperature, 35 ml of 0.26 M sodium methanolate inmethanol were added. After 1 h, 35 ml of methanol and 35 ml of 2 Nsulphuric acid were added, and the reaction mixture was stirred foranother 15 min, neutralized to pH 6.0 with saturated KHCO₃ (28 ml) andextracted twice with ethyl acetate. The organic phase was washed 2 timeswith saturated NaCl, dried over anhydrous Na₂SO₄ and filtered.Subsequently, solvent was evaporated under reduced pressure. The residuewas chromatographed on silica gel (eluent: 5:1 ethylacetate/methanol).

7.3 g ofH-MeLeu-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(OAc)-Abu-Sar-OMewere obtained (yield: 76%).

HPLC tr=268.23 nm (98%)

ES/MS: m/z: 1277.5 [M+H⁺], 639.2 [M+2H⁺]

Synthesis ofH-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(OAc)-Abu-Sar-OMe

DMAP (2.3 mmoles; 334 mg) and phenylisothiocyanate (6.9 mmoles; 0.75 ml)were added to a solution ofH-MeLeu-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(OAc)-Abu-Sar-OMe(4.6 mmoles; 7 g) in 48 ml tetrahydrofuran. After 2 h, solvant wasevaporated, and the crude product was chromatographed on silica gel(eluants: 9:1 tert-butyl methyl ether (MTBE)/ethylacetate (1);

9:1 MTBE/methanol (2)). 5.8 g of Ph-NH—C(S)—MeLeu-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(OAc)-Abu-Sar-OMe wereobtained (90% yield).

13.8 ml trifluororacetic acid were added to a solution of the lattercompound (4 mmoles; 5.6 g) in 290 ml dichloromethane. After 1 h ofreaction, the mixture was neutralized using KHCO₃ and diluted with 500ml dichloromethane. The organic phase was washed 2 times with saturatedNaCl, dried over anhydrous Na₂SO₄ and filtered. Subsequently, solventwas evaporated under reduced pressure. The residue was chromatographedon silica gel (eluants: 9:1 MTBE/ethylacetate (1); 3:1 MTBE/methanol(2)). 2.8 gH-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(OAc)-Abu-Sar-OMe wereobtained (61% yield).

HPLC tr=25.80 nm (99%)

ES/MS: m/z: 1050.5 [M+H⁺], 547.7 [M+2H+]

Synthesis ofBoc-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(OAc)-Abu-NMe-CH₂—CH₂—OH

Fluoro-N,N,N′,-tetramethylformamidinium hexafluorophosphate (TFFH) (0.96mmoles; 0.25 g) was added, under an inert atmosphere, to a solution ofH-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(OAc)-Abu-Sar-OMe (0.87mmoles; 1.00 g), DIPEA (2.78 mmoles; 0.48 ml) and Boc-D-MeAla-EtVal-OH(0.96 mmoles; 0.32 g) in 15 ml dichloromethane. After 15 min,dichloromethane was evaporated, and the residue was taken up inethylacetate. The organic phase was washed successively with a saturatedNaHCO₃ solution, a 10% solution of citric acid and a saturated NaClsolution, and was then dried over anhydrous Na₂SO₄ and concentrated.Chromatography on silica gel (in 98:2 ethylacetate/methanol) yielded1.14 g (90%)Boc-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(OAc)-Abu-Sar-OMe.

The latter product (0.64 mmoles; 0.93 g) was taken up in 45 ml anhydrousmethanol, and sodium borohydride (25.5 mmoles; 0.96 g) was added insmall portions at 15-min intervals over a period of 3 h 30 min. At 4 h,the reaction mixture was cooled to 0° C., hydrolysed by addition of 10%citric acid and concentrated. Residue was taken up in ethylacetate. Theorganic phase was washed with a 10% solution of citric acid and asaturated NaCl solution, and was then dried over anhydrous Na₂SO₄ andconcentrated. After chromatography on silica gel (in 95:5ethylacetate/methanol) 0.63 g (81%) ofBoc-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(OAc)-Abu-NMe-CH₂—CH₂—OHwere obtained.

ES/MS: m/z: 1434.9 [M+H⁺], 717.9 [M+2H⁺]

Synthesis ofH-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt-Abu-OH

Methanesulfonic acid (3.18 mmoles; 2.060 ml) was added to a solution ofBoc-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(OAc)-Abu-NMe-CH₂—CH₂—OH(0.425 mmoles; 610 mg) in 42.5 ml methanol, and the mixture was heatedto and maintained at 50° C. Progress of the reaction was monitored byHPLC and mass spectrometry. After 80 h, the mixture was cooled to 0° C.,and hydrolysed by addition of 1 M NaHCO₃. Methanol was eliminated, andthe residue was taken up in ethylacetate. The organic phase was washedwith 1 M NaHCO₃ and then a saturated NaCl solution, dried over anhydrousNa₂SO₄ and concentrated. The product (557 mg),H-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(OAc)-Abu-O—CH₂—CH₂—NHMe,was used in the next step without purification.

Product (0.42 mmoles; 557 mg) was dissolved in 20 ml methanol andcombined, under an inert atmosphere, with a solution of sodiummethanolate (1.26 mmoles) in 1.26 ml methanol. After 18 h at roomtemperature, the reaction mixture was cooled to 0° C., and sodiumhydroxide (4.2 mmoles; 168 mg) in 5 ml water was added dropwise. After21 h at room temperature, the reaction mixture was again cooled to 0° C.and neutralized with 1 M KHSO₄. Methanol was eliminated, and the residuewas dissolved in ethylacetate. The organic phase was washed with asemi-saturated NaCl solution, dried over anhydrous Na₂SO₄ andconcentrated. The product (335 mg; 64%),H-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt-Abu-OH, wasused in the next step without purification.

HPLC tr=26.27 nm (86%)

ES/MS: m/z: 1235.5 [M+H⁺], 618.2 [M+2H⁺]

Synthesis of [D-MeAla]3-[EtVal]4-CsA

Under an inert atmosphere, a solution ofH-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt-Abu-OH(0.162 mmoles; 200 mg) and sym.collidine (1.78 mmoles; 0.24 ml) in 50 mldichloromethane was added dropwise to a solution of(7-azabenzotriazole-1-yloxy)tripyrrolidinophosphoniumhexafluoro-phosphate (PyAOP, 0.486 mmoles; 254 mg) in 3.2 literdichloromethane. 72 h later, the reaction mixture was hydrolysed byaddition of a 10% Na₂CO₃ solution. Dichloromethane was evaporated, andresidue taken up in ethylacetate. The organic phase was washedsuccessively with a 0.1 N HCl solution and a saturated solution of NaCl,dried over anhydrous Na₂SO₄ and concentrated. Crude product was purifiedon silica gel, yielding 110 mg (59%) [D-MeAla]³-[EtVal]⁴-CsA HPLCtr=30.54 nm (100%)

ES/MS: m/z: 1217.6 [M+H⁺], 609.3 [M+2H+]

EXAMPLE 2 Oral Formulations of Cyclosporins of the Invention

Amounts are expressed as % w/w.

Example A

Cyclosporin of the invention 10 Glycofurol 75 35.95 Miglycol 812 18Cremophor RH40 35.95 Alpha-Tocopherol 0.1

Example B

Cyclosporin of the invention 10 Tetraglycol 2 Captex 800 2 Nikkol HCO-4085.9 Butylhydroxytoluene (BHT) 0.1

Example C

Cyclosporin of the invention 10 Glycofurol 75 39.95 Miglycol 812 14Cremophor RH40 36 Butylhydroxyanisole (BHA) 0.05-0.1

Example D

Cyclosporin of the invention 10 Tetraglycol 10 Myritol 5 Cremophor RH4074.9 Alpha-Tocopherol 0.1

Example E

Cyclosporin of the invention 10 Ethanol 9 Propylene glycol 8 CremophorRH40 41 Glycerol monolinoleate 32

For individual components of formulations A-D and for methods ofpreparation see British Patent Appl. No. 2,222,770.

1. A method of treating of hepatitis C infection in a subject comprisingadministering to the subject [D-MeAla]³-[EtVal]⁴-CsA in combination withone or both of an interferon alpha and ribavirin as part of a doseregimen designed to obtain the benefits of the combination.
 2. Themethod of claim 1 wherein [D-MeAla]³-[EtVal]⁴-CsA and one or both of aninterferon alpha and ribavirin are administered together as part of asingle composition or are administered separately.
 3. The method ofclaim 1 wherein [D-MeAla]³-[EtVal]⁴-CsA and one or both of an interferonalpha and ribavirin are administered simultaneously or in sequence.
 4. Amethod of treating hepatitis C infection in a subject comprisingadministering to the subject effective amounts of[D-MeAla]³-[EtVal]⁴-CsA and one or both of an interferon alpha andribavirin.
 5. The method of claim 4 wherein the interferon alpha orribavirin are administered at doses that are less than or equal to thedoses at which they are effective as single therapeutic agents.
 6. Themethod of claim 4 wherein [D-MeAla]³-[EtVal]⁴-CsA and one or both of aninterferon alpha and ribavirin are administered together as part of asingle composition or are administered separately.
 7. The method ofclaim 4 wherein [D-MeAla]³-[EtVal]⁴-CsA and one or both of an interferonalpha and ribavirin are administered simultaneously or in sequence.